Structural genes encoding the various allelic and maturation forms of preprothaumatin and mutations thereof, recombinant cloning vehicles comprising said structural genes and expression thereof in transformed microbial host cells

Claim 1

A process for producing DNA sequences encoding non-processed and partly processed preprothaumatin and the various allelic forms of the preprothaumatin gene, characterized in that: (1) thaumatin messenger RNA-containing material is isolated from arils of Thaumatococcus daniellii; (2) said thaumatin messenger RNA-containing material is purified; (3) the thaumatin messenger RNA is converted into single-stranded DNA using a reverse transcriptase and subsequently to double-stranded DNA by a DNA polymerase; (4) said double-stranded DNA is integrated in plasmids; (5) said plasmids are transferred into bacterial cells and hybrid plasmid DNA molecules are selected to yield a plasmid containing a substantially complete copy of thaumatin mRNA; (6) said plasmids are treated with a series of restriction endonucleases to obtain a fragment A according to Fig. 10 and a fragment B according to Fig. 11 which are ligated and integrated in a microbial plasmid; (7) cloning an appropriate fragment of said plasmid and producing a single-stranded DNA, which is subsequently used for complementary DNA synthesis in the presence of an appropriate primer; (8) using the complementary DNA strand obtained by denaturation of the double-stranded DNA, as a template for DNA synthesis in the presence of an appropriate primer and obtain the prethaumatin nucleotide sequence 32-718 (fig. 4) and the prothaumatin sequence 98-736 (fig. 3) and (9) optionally introducing mutations in said sequences.